Technical Field
[0001] The present invention relates to human monoclonal antibodies (hereinafter abbreviated
to MCA) against cytomegalovirus (hereinafter abbreviated to CMV) and a process for
producing the same. The object of this invention is to provide human MCA, specific
to CMV, which are useful for the diagnosis, prevention, and therapy of CMV infectious
diseases.
Background Art
[0002] CMV is one of the viruses which belong to the herpesvirus group and is composed of
DNA, core protein, capsid, and envelope. Scarcely and diseases are caused in the generality
of cases even when a human being is infected by this virus; however, it sometimes
causes fatal infections such as hepatitis seen with an infected newborn who has low
immunity and interstitial pneumania found with a patient who is immunosuppressed because
of organ transplantation. Accordingly, effective means of diagnosis, prevention, and
therapy of such infections have been greatly needed in medical circles. Condie et
al. (see American J. Medicine, March 30, 1984, pp. 134-141) report succeful cases
where administration of human serum immunoglobulin with a high antibody titer to CMV
protected bone marrow transplant recipients from CMV infections and interstitial penumonia
arising from the same. Human serum immunoglobulin with a high titer is prepared by
collecting and fractionating high-titer blood plasma only, the selection of which
is made by checking the antibody titer of the serum of donors beforehand. The antibody
titer to CMV of serum immunoglobulin thus prepared is at the most 10 times that of
serum immunoglobulin prepared from blood plasma collected randomly and collection
of such high-titer serum immunoglobulin as this is very difficult to make in an enough
amount for stabilized supply.
[0003] The production of high-purity antibody, or MCA, has been made possible since the
establishment of the hybridoma method by Milstein and Köhler. Rasmussen et al. (see
Proc. Natl. Acad. Sci., U.S.A., 81, pp. 876-880, 1984) provided hybridomas producible
of MCA specific to CMV by fusing mouse myeloma cells and spleen cells of mice immunized
with CMV. Several research groups have, besides her, obtained mouse MCA specific to
CMV (for instance, Goldstein et al., in Infection and Immunity, 38, pp. 273-281, 1982;
Pereira et al., in Infection and Immunity, 36, pp. 924-932, 1982). Of these MCA, few
MCA are seen to have enough active capacity to neutralize viruses; however, MCA provided
by Rasmussen et al. has a capacity to neutralize CMV at about 10 pg/ml. This neutralizing
capacity is very high when compared with high-titer serum immunoglobulin and is expected
to be of use for the prevention or remedy of CMV infections. But, being derived from
mice, these MCA are recognized as a foreign substance upon administration to humans,
thus causing harmful side effects. Therefore, the development of anti-CMV MCA, arising
from humans instead of mice, are hoped for.
[0004] Human MCA are generally produced from hybridomas obtained by cell fusion between
mouse myeloma cells, human myeloma cells, or cells established from other lymphoid
tissues and human lymphocytes. They are also produced from lymphoblast cells obtained
by transforming human lymphocytes by Epstein
-Barr virus. Many attempts have been made since 1980 to develop human MCA but every
method has had its own characteristic problem. Production of MCA from hybridoma obtained
by cell fusion of mouse myeloma cells with human lymphocytes is not stable, while
the cell fusion between human myeloma cells and human lymphocytes is very low in efficiency.
Production of MCA from lymphoblast cells obtained by means of EB virus involves problems
of low yield and instability. Moreover, EB virus is capable of causing tumors, thus
offering a serious problem of safety. The technics of establishing MCA-producing cells
involves great difficulties as described in the above; however, there is another serious
barrier we find lying in the means of obtaining MCA specific to specialantigens. It
is a problem generally seen in collecting lymphocytes from fully immunized humans.
Generally speaking, lymphocytes of normal persons are in many cases sensitized by
CMV but its degree of immunity is very low. Therefore, even when hybridomas or lymphoblast
cells are established from lymphocytes of normal persons, it is hardly expected to
obtain cells producible of anti-CMV MCA.
[0005] With regard to human MCA against CMV, a report is made on a single case of establishing
a cell line producing the same (see J. Immunol., 133, pp. 2202-2205). According to
this report, a MCA-producing cell line is obtained by transforming lymphocytes of
normal persons with EB virus. The report, however, contains only one photograph of
fluorescence antibody and it is not made clear whether a cell line which produces
MCA with secured stability is established or not. Morever, it also accompanies a problem
of carcinogenecity arising from EB virus as mentioned before and it is apprehended
that it may involve a great risk of life upon its in vivo administration. And it is
made apparent too that this MCA is incapable of neutralizing CMV even if it binds
to CMV.
[0006] As explained in the above, the hardest problem encountered in efficiently obtaining
desired specific human MCA is the difficulty in sufficiently immunizing human lymphocytes
with a specific antigen. In a case where mice are used, even an antigen which is harmful
in vivo can be given to them and immunization can also be carried out according to
a schedule conveniently suited for the purpose. This is impracticable in the case
of human beings, since CMV a pathogen and so far as no vaccine has yet been developed,
it is not allowed morally to give CMV to human beings intentionally for the purpose
of immunization. Another problem that confronts the productionof human MCA stably.
Both hybridoma method and EB virus method have merits and demerits as described in
the above.
Disclosure of the Invention
[0007] As the result of an intensive research conducted with the object of obtaining anti-CMV
human MCA, the present inventors have succeeded in obtaining hybridomas producible
of anti-CMV human MCA by a method in which human lymphocytes in vitro sensitized to
CMV or protein or glycoprotein arising from CMV and mouse myeloma cells (in the presence
of mitogen are subjected to cell fusion. Hybridomas thus obtained and/or a cell line
arising therefrom are then cultured and anti-CMV human MCA are obtained from the culture
supernatant.
[0008] Human MCA of this invention is an antibody which reacts to CMV and/or CMV-infected
cells. Of the anti-CMV human MCA of this invention, desirable ones are IgG 1 type,
which are further classified into two kinds. One kind of MCA recognizes CMV antigen
protein with a molecular weight of about 64,000 or plural CMV antigen proteins having
antigen protein with a molecular weight of about 64,000 as the chief element. Another
kind of MCA recognizes CMV antigen protein with a molecular weight of about 130,000
and one with a molecular weight of about 55,000. The latter is capable of neutralizing
CMV and is produced from hybridoma C41 which is deposited with American Type Culture
Collection (ATCC) under deposit number HP 9215 or any other similar hybridomas which
recognize antigen protein.
Brief Description of Drawings
[0009] Fig. 1 is a drawing which shows the neutralization activity of anti-CMV human MCA
of this invention against CMV. Fig. 2 is a drawing to show the result of immunoprecipitation
assay of anti-CMV human MCA of this invention.
Best Mode of Carrying Out the Invention
[0010] Human lymphocytes which are used in this invention are found contained in the spleen,
lymph node, peripheral blood, bone-marrow, tonsils, and adenoids. Lymphocytes of any
source may be used to secure the object of this invention; however, those obtained
from the spleen, bone-marrow, and tonsils are preferable.
[0011] As the mouse myeloma cells, 8-azaguanine resistant cell lines are advantageously
used. There are P3X65Ag8 cell line, P3-NSl/l-Ag4-1 cell line, P3X63AgUl cell line,
SP2/OAgl4 cell line, P3X63Ag8.6.5.3 cell line, PMC11-45.6.TG1.7 cell line, and SP-1
cell line of BALB/C mouse among publicly known ones.
[0012] In this invention, human lymphocytes are sensitized to antigen in vitro in the presence
of mitogen prior to the cell fusion between the human lymphocytes and mouse myeloma
cells is performed. In the case of human beings, though there exists lymphocytes producible
of antibodies against CMV in normal persons, they are very small in number. There
is accordingly very little possibility of obtaining the desired hybridomas. On the
contrary, when the method of this invention, in which human lymphocytes are sensitized
to an antigen in vitro, is adopted, the lymphocytes, which produce antibody specific
to the antigen, start selective differentiation and proliferation and consequently
make it possible to obtain the desired MCA-producing hybridomas efficiently.
[0013] In the Examples of this invention, AD 169 strain of CMV is used as the sensitizing
antigen but other CMV strains for experimental use or clinically separated CMV strains
can also be used for sensitization. Instead of CMV itself, its constituent proteins
or constituent glycoproteins may also be used.
[0014] Any type of mitogen may be used so far as it can promote the differentiation and
proliferation of lymphocytes and to give its examples, there are pokeweed mitogen
(PWM), B cell growth factor (BCGF), protein A, phytohemagglutinin (PHA), and concanavalin
A. Desirable ones are 2-200 pg/ml PWM and 1/100-1/3 volume BCGF.
[0015] No limit is specially set to the method and conditions of sensitization; however,
advisable conditions include the antigen (CMV, or a protein or a glycoprotein arising
from CMV) concentration of 1 µg/ml - 1 ng/ml, PWM concentration of 2-200 pg/ml, BCGF
concentration of 1/100-1/3 volume, and lymphocytes (antibody-producing cells) concentration
of 1X10
5-1X10
7 cells/ml. It is advisable to keep the culture temperature at 35-40°C and culture
time at 4-10 days, preferably 6-8 days. Any liquid culture medium may be used so far
as it contains serum of human being, bovine, equine, etc.; however, a liquid culture
medium (such as RPMI 1640) which contains fetal calf serum (FCS) is especially preferable.
[0016] Thus obtained antibody-producing human cells sensitized to CMV and mouse myeloma
cells are then subjected to cell fusion according to a publicly known method. For
instance, lymphocytes and myeloma cells are mixed at a ratio of 10:1-1:100, preferably
1:1-1:10 and a suitable cell fusion solution such as RPMI 1640 which contains about
35% polyethylene glycol (molecular weight about 1,000-6,000) and about 7.5% dimethyl
sulfoxide is added to the mixture. After the mixture is stirred at room temperature
- 37°C for 1 to several minutes, it is diluted little by little with RPMI 1640 containing
10% ECS. After washing, the mixture is adjusted with HAT (hypoxanthine-aminopterin-thymidine)
selective medium to have a cell concentration of 1-5X10
5 cells/ml. The cells were placed in 0.2 ml portions in a 96-well culture plate to
be cultured in 5% C0
2 air at 35-38°C for 3-4 weeks. In HAT liquid culture medium only hybridomas survive
and 8-azaguanine resistant myeloma cells and fused cells between myelomas can not
survive (unfused antibody producing cells perish in a course of several days).
[0017] After the culture is over, the antibody titer in each liquid culture medium is measured
and only such hybridomas that produce antibodies as required are selected and isolated
(cloning). The measurement of antibody titer in the liquid culture medium can be made
by such a method in which the binding of antibody to antigen is checked as radioimmunoassay
(RIA), enzyme- linked immunosorbent assay (ELISA), and fluorescent antibody technique
and also by a method in which the antibody activity that can hinder the biological
activity of virus is measured. Mouse-human hybridoma producible of anti-CMV human
MCA of this invention can be freeze-stored. When this hybridoma cell line and/or cell
line arising therefrom is mass-cultured by a proper method, the desired human MCA
of this invention can be obtained from the culture supernatant. Also, this hybridoma
can be transplanted to animals to cause tumor and human MCA may be obtained from their
abdominal dropsy and serum.
[0018] Anti-CMV human MCA thus obtained has the following chracteristic properties. The
anti-CMV human MCA obtained by this invention reacts commonly to many kinds of experimental
CMV strains and clinically separated strains but does not react to other herpes group
including herpes simples virus, EB virus, and chickenpox herpes zoster virus. It reacts
to CMV-infected cells but not to uninfected cells. Therefore, it may be said that
these MCA are specific to CMV. Human MCA which have thus been clearly pointed out
to the specific to CMV have for the first time come to be made obtainable by this
invention.
[0019] CMV are composed of many antigenic substances. As the result of study made to clarify
what CMV constituents the human MCA of this invention react to, it has been made clear
that MCA of the first group react to proteins (inclusive of glycoproteins; the same
shall apply hereinafter) having a molecular weight of about 64,000 and several number
of antigen proteins mainly consisting of proteins having a molecular weight of about
64,000. These MCA have no ability to neutralize virus. While MCA of the second group
react to proteins having molecular weight of about 130,000 and proteins having molecular
weight of about 55,000 and it has been clarified that some of MCA belonging to this
group have a strong activity of neutralizing virus. Judging from these properties,
MCA belonging to the first group and those belonging to the second group can be used
for the diagnosis of CMV infection, while MCA belonging to the second group can be
advantageously used for the prevention and therapy of CMV infection.
[0020] In this invention, no limit is set forth as to antigenic substances and all human
MCA that are specific to CMV are involved in the scope of this invention. It is, however,
a very important property for MCA to have an activity of neutralizing (inactivating)
virus when judged from the viewpoint of prevention and therapy.
[0021] The present invention is described in detail by the following examples.
Example 1
(1) Preparation of CMV antigen
[0022] HEL cells grown in monolayers were mixed with cells infected by AD 169 strain at
a ratio of 7:1 and cultured for 5 days in a C0
2 incubator. The infected cells thus obtained were peeled off with phosphate buffer
saline containing 0.1% EDTA and collected. The pooled substances were then subjected
to ultrasonic breaking for.45 seconds and centrifuged at 3,000 rpm for 20 minutes
to obtain supernatant, which was placed on a double- layered sucrose solution, one
layer being 66% and the other 20%, and centrifuged at 10,000 X g for 1 hour. Coming
together between the 60% layer and the 20% layer by nature, the viruses were collected
and dispersed thoroughly by ultrasonic treatment. The dispersion liquid was placed
on a CsC1
2 solution of 1.28g/cm
3 and centrifuged at 100,000 X g for 42 hours. As the result, CMV were found gathering
in the CsC1
2 solution at a density of 1.29g/cm
3 where a density gradient was generated and collected for use as an antigen for in
vitro sensitization.
[0023] Also, antigen for ELISA use were prepared as follows. HEL cells infected with AD
169 were cultured in a C0
2 incubator and infected cells were collected when all the cells were observed having
developed cell denaturation. These cells were ultrasonically treated for 3 minutes
and centrifuged at 3,000 rpm for 20 minutes to obtain the supernatant. This was used
as an antigen for ELISA use.
(2) Sensitization of lymphocytes with CMV
[0024] Human spleen lymphocytes were suspended in a liquid culture medium A (RPMI 1640+20%
fetal calf serum + 20mM HEPES + 2mM glutamine + lmM Na pyruvic acid + 0.02mg/ml serine
+ 80 pg/ml gentamicin), whose cell density was 12X10
5 cells/ml. This cell suspension was placed in 15 wells of a 24-well culture plate
in portions of 1 ml. They were divided into 5 groups of 3 wells, and the first group
had nothing added thereto, the second group had CMV antigen 12 ng protein/ml added
thereto, the third group had 10 ml of BCGF, the fourth group had the same amount of
CMV and BCGF, and the fifth group had CMV and 20 µg/ml of PWM. The culture plate was
incubated at 37°C in 5% CO
2 air for 6 days.
(3) Cell fusion with mouse myeloma cells, P3X63Ag 8U1 cell line (abbreviated to P3Ul)
[0025] P3U1 had been cultured beforehand in a liquid culture medium B (RPMI 1640 + 10% fetal
calf serum + 2mM glutamine + 80 µg/ml gentamicin). The cell density at the time of
its use was 6X10
5 cells/ml. Five groups (of 3 wells) of sensitized lymphocytes obtained in the preceding
(2) and P3Ul were separately washed twice with serum-free RPMI 1640. Lymphocytes in
the 3 wells of the respective groups and 5Xl0
6 cells of P3U1 were placed together in test tubes. They were centrifuged at 1,500
rpm for 5 minutes and the supernatants were discarded. Cell pellets were dispersed
thoroughly by tapping the test tubes. 0.5 ml of polyethylene glycol solution (RPMI
1640 5.75 ml + polyethylene glycol 100 3.5 ml + dimethyl sulfoxide 0.75 ml) abbreviated
to PEG solution) was added to each test tube allowing cells to suspend in the solution
gently. One minute later 0.5 ml of RPMI 1640, another 1 minute later 1 ml of RPMI
1640, another 2 minutes later 4 ml of HAT liquid culture medium (RPMI 1640 + 20% fetal
calf serum + 80 µg/ml of gentamicin + 95 µM hypoxanthine + 0.4 µM aminopterin + 1.6
µM thymidine), and another 2 minutes later 4 ml of
HAT liquid culture medium were added, thus finally making up a total of 25 ml of cell
suspending solution. This solution was seeded in a culture plate (96 wells) and cultured
at 37°C in 5% C0
2 air. Half of the medium was exchanged for a fresh HT culture medium (omitting A from
HAT medium) every week repeatedly, thus obtaining hybridoma.
(4) Measurement of human IgG and CMV antibody
[0026] The measurement was made by ELISA. For making the measurement of human IgG, goat
anti-human IgG antibody (10 µg/ml), and for making the measurement of anti-CMV antibody,
2 µg protein/ml of CMV (AD 169 strain) were respectively coated to Falcon microtest
III 96-well plates in portions of 50 ul/well. 60 µl of hybridoma culture supernatant
was added to the plates and left standing at room temperature for 1 hour. After having
been washed three times with Hank's balanced salt solution buffer (HBSS-B) containg
1% bovine serum albumin (BSA), the plates had 60 µl of anti-goat human IgG antibody-alkaline
phosphatase (2000-fold diluted solution) added and were made to go through reaction
at room temperature for 1 hour. The plates were again washed 3 times with HBSS-B and
then 100 µl of a solution prepared by dissolving P-nitrophenylphosphate in 1 M diethanolamine
+ 1 mM MgC1
2 solution of pH 9.5 at a ratio of 0.6 mg/ml was added thereto. About 30 to 60 minutes
thereafter, the absorbance at 405 nm was measured and the required values were calculated
by comparing the measurement with that of standard IgG solution or standard CMV positive
serum.
[0027] All the groups had their cells seeded to the respective 96-well plates and the number
of wells in which hybridomas were generated (observed by the naked eye), furthermore,
the number of wells in which said hybridomas had produced human IgG, and the number
of wells in which anti-CMV antibodies had been produced were counted and shown in
Table 1. When CMV and BCGF were added, the largest number of anti-CMV antibody-producing
hybridomas were generated.
Example 2
[0028] Human spleen lymphocytes were sensitized with CMV or PWM according to Example 1,
(2), and cell-fused with P3Ul according to Example 1, (3). Of the hybridomas thus
obtained, those hybridomas which had produced MCA were subjected to cloning as follows.
(1) Cloning of hybridomas
[0029] Cloning was performed by limiting dilution. Cells were taken out of anti-CMV antibody
positive wells and were seeded in portions of 1 cell/well or 10 cells/well in the
respective well containing a liquid culture medium B. Cells were found grown proliferously
enough 2 weeks later, and it was checked by ELISA if there were anti-CMV.MCA in the
supernatants, thus selecting hybridomas which were producible of anti-CMV.MCA.
[0030] In this way, hybridomas Cl, C3, C4, C7, C23 and C41 were established. These hybridomas
are since continuing stably to produce anti-CMV.human MCA of IgG type. Hybridoma C41
was diposited with American Type Culture Collection (ATCC) on September 29, 1986,
under deposit number HB 9215.
(2) Preparation of anti-CMV.human MCA
[0031] C41, one of the hybridomas obtained in the above, was cultured in a serum-free ITES
culture medium (RPMI 1640 2 volumes + Dulbecco's MEM 1 volume + F12 1 volume + insulin
8.5 µg/ml + transferrin 2 pg/ml + ethanolamine 2 µM + selenite 2.5X10-
8M). Its culture supernatant was concentrated by ultrafiltration (Amicon P30) and dialyzed
against 0.02 M sodium phosphate (pH 7.8). The dialyzate was put to DE 52 column (PHarmacia)
equilibrated with the same buffer and recovered human MCA from unadsorbed fractions.
Upon analysis by sodium dodecyl culfate-polyacrylamide gel electrophoresis (SDS-PAGE),
it was confirmed to be a parified MCA preparation comprising H-chain and L-chain.
Example 3
(1) Reactivity of anti-CMV.human MCA to infected cells
[0032] Anti-CMV.human MCA Cl, C3, C4, C7, C23 and C41 were made to react to HEL cells infected
with AD 169 strain cf CMV or Hi-1 strain and to those not infected cells. Further,
fluorescein isothiocyanate (FITC)-labeled human IgG antibodies were made to react
to them and were observed under a fluorescence microscope. The result showed that
all MCA did not react to the uninfected cells but reacted only to infected cells.
It was also found that Cl, C3, C4, and C7 reacted to CMV antigen in the cytoplasm
of cells infected with AD 169 or Hi-1 but did not reacted to CMV antigen on their
cell membrane. On the other hand, C23 and C41 reacted to both CMV antigen in the cytoplasm
and CMV antigen on the cell membrane.
(2) Reactivity of anti-CMV.human MCA to solubilized virus antigen
[0033] With the object of studying the binding activity of anti-CMV.MCA to herpesviruses,
solubilized antigens were prepared according to Example 1, (1), from HEL cells infected
respectively with 2 strains of HSV-1, 2 strains of HSV-2, 2 strains of VZV, 6 strains
of CMV, and 1 strain of EBV and unifected HEL cells, and ELISA was performed with
them according to Example 1, (4). The result is shown in Table 2.
[0034] Anti-CMV.MCA (Cl, C2, C3, C7, C23, and C41) were all found to bind to CMV only but
not to bind to other herpesvirus and host cells. Anti-CMV·MCA, except for C41, were
found to bind to all the 6 strains of CMV. Incidentally, Hl is human MCA against herpes
simplex virus (HSV).
Example 4
Virus neutralizing activity of anti-CMV. human MCA
[0035] A mixture of 200 µl of human MCA solution, 100 µl of 5-fold diluted solution of 200
CH50/ml of fresh guinea pig serum, and 100 µl of solution containing 737 pfa (plaque-forming
units) of CMV (AD 169 strain) was incubated at 37°C for 1 hour. After 100 µl of mixed
solution was spread over HEL cells incubated in a 6-well plate and again incubated
at 37°C for 1 hour, MEM medium which contained 0.5% (W/V) agarose and 5% (V/V) FCS
was added thereto and incubated for 11 days in a C0
2 incubator. The resulting single-layer cells were fixed with a 10% formalin solution
and stained with a 0.3% Methylene Blue solution to determine the plaque-forming units
(pfu) of CMV-infected cells. The neutralizing activity was calculated as follows.
[0036] The result is shown in Fig. 1. 50% neutralization was indicated by C23 at 0.75 pg/ml
and by C41 at 0.18 pg/ml; however, Cl, C3, C4, and C7 showed no neutralization activity.
In case where Hi-1 strain was used, C23 showed 10% neutralization at 3.3 µg/ml and
C41 showed 100% neutralization at 0.95 µg/ml, while C3 showed only 35% neutralization
at 17.7 µgml.
[0037] Upon determination of neutralizing activity of 5 strains of CMV strains excepting
AD 169 strain, C23 showed 95% or more of neutralization for all virus strains at 10
µg/ml. While C41 showed, corresponding closely to the ELISA data given in Table 2
of Example 3, (2), somewhat low neutralizing activity of 20% and 64% to No.12 strain
and YAN-3 strain respectively and more than 80% neutralizing activity to other strains.
Example 5
Immunoprecipitation analysis of anti-CMV· human MCA
[0038] Immunoprecipitation analysis was conducted to determine what viral constituent(s)
those human MCA react with. HEL cells wer infected with CMV (AD 169 strain) and isotope-labeled
with 35S-methionine. The labeled cells were dissolved in a solution comprising 0.01M
Tris. HC1 + 0.15 M NaCl + 1% sodium deoxycholate + 1% Triton X 100 + 0.1% sodium dodecyl
sulfate (SDS) + 1 mM phnyl methyl sulfonyl fluoride (pH 7.4). Anti-CMV·human MCA was
added thereto to generate an antigen-antibody complex which was then subjected to
adsorption purification by use of protein A - Sepharose 4B. The purified product was
heat-treated at 100°C for 3 minutes in a solution of 0.125 M Tris·HCl + 1% SDS + 3%
2-mercaptoethanol + 15% glycerin (pH 8.2) and the resulting supernatant was subjected
to SDS- polyacrylamide gel electrophoresis. After the electrophoresis was over, the
gel was dried and exposed to X-ray film at -70°C.
[0039] The result is shown in Fig. 2. It has been found that Cl reacts with virus antigens
having molecular weights of about 64,000 and about 50,000, C2 reacts with the ones
having molecular weights of about 64,000 and about 46,000 C4 and C7 react with the
one having a molecular weight of about 64,000, and C23 and C41 react with the ones
having molecular weights of about 130,000 and about 55,000.
Example 6
[0040] Isotypical identification of anti-CMV.human MCA
[0041] Isotypical identification of human MCA was carried out as follows. Identification
of H-chain was made by the immunodiffusion test (Ouchterlony test) with the use of
rabbit antiserum against human IgGl, IgG2, IgG3, and IgG 4. Identification of L-chain
was conducted by ELISA in which AD169-infected cells were used as antigen plate and
alkaline phosphatase-labeled goat anti-human K-chain or λ-chain was used as secondary
antibody.
[0042] The result is shown in Table 3.
[0043] Industrial Applications
[0044] Anti-CMV.human MCA of the present invention can be advantageously used as a diagnostic
for CMV infections and also as a preventive and a remedy for CMV infectious diseases.